Abstract
The renal proximal tubule (PT) is the major target of cadmium (Cd2+) toxicity where Cd2+ causes stress and apoptosis. Autophagy is induced by cell stress, e.g., endoplasmic reticulum (ER) stress, and may contribute to cell survival or death. The role of autophagy in Cd2+-induced nephrotoxicity remains unsettled due to contradictory results and lack of evidence for autophagic machinery damage by Cd2+. Cd2+-induced autophagy in rat kidney PT cell line NRK-52E and its role in cell death was investigated. Increased LC3-II and decreased p62 as autophagy markers indicate rapid induction of autophagic flux by Cd2+ (5–10 µM) after 1 h, accompanied by ER stress (increased p-PERK, p-eIF2α, CHOP). Cd2+ exposure exceeding 3 h results in p62/LC3-II accumulation, but diminished effect of lysosomal inhibitors (bafilomycin A1, pepstatin A +E-64d) on p62/LC3-II levels, indicating decreased autophagic flux and cargo degradation. At 24 h exposure, Cd2+ (5–25 µM) activates intrinsic apoptotic pathways (Bax/Bcl-2, PARP-1), which is not evident earlier (≤6 h) although cell viability by MTT assay is decreased. Autophagy inducer rapamycin (100 nM) does not overcome autophagy inhibition or Cd2+-induced cell viability loss. The autophagosome–lysosome fusion inhibitor liensinine (5 μM) increases CHOP and Bax/Bcl-2-dependent apoptosis by low Cd2+ stress, but not by high Cd2+. Lysosomal instability by Cd2+ (5 μM; 6 h) is indicated by increases in cellular sphingomyelin and membrane fluidity and decreases in cathepsins and LAMP1. The data suggest dual and temporal impact of Cd2+ on autophagy: Low Cd2+ stress rapidly activates autophagy counteracting damage but Cd2+ stress accrual disrupts autophagic flux and lysosomal stability, possibly resulting in lysosomal cell death.
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Funding was obtained from the Center for Biomedical Training and Research (ZBAF) of the University of Witten/Herdecke (F.T.), Deutsche Forschungsgemeinschaft (TH345/11-1 to F.T.), BMBF 01DN16039 (F.T.) Max Kade Foundation (W.-K.L.), CONACYT (Consejo Nacional de Ciencia y Tecnología, Mexico) (235537 to M.P.S.-S.), NSERC Discovery grant (E.J.P.) and a fellowship from PhD program Biomedicine, Witten/Herdecke University (W. A.-H.).
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W.-K. Lee, S. Probst and M. P. Santoyo-Sánchez contributed equally.
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Lee, WK., Probst, S., Santoyo-Sánchez, M.P. et al. Initial autophagic protection switches to disruption of autophagic flux by lysosomal instability during cadmium stress accrual in renal NRK-52E cells. Arch Toxicol 91, 3225–3245 (2017). https://doi.org/10.1007/s00204-017-1942-9
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DOI: https://doi.org/10.1007/s00204-017-1942-9